The PD-L1/PD-1 immune checkpoint axis is the strongest T cell exhaustion inducer. As immune dysfunction occurs during obesity, we analyzed the impact of obesity on PD-L1/PD-1 expression in white adipose tissue (WAT) in mice and in human white adipocytes. We found that PD-L1 was overexpressed in WAT of diet-induced obese mice and was associated with increased expression of PD-1 in visceral but not subcutaneous WAT. Human in vitro cocultures with adipose-tissue-derived mesenchymal stem cells (ASC) and mononuclear cells demonstrated that the presence of ASC harvested from obese WAT (i) enhanced PD-L1 expression as compared with ASC from lean WAT, (ii) decreased Th1 cell cytokine secretion, and (iii) resulted in decreased cytolytic activity towards adipocytes. Moreover, (iv) the implication of PD-L1 in obese ASC-mediated T cell dysfunction was demonstrated through PD-L1 blockade. Finally, (v) conditioned media gathered from these cocultures enhanced PD-L1 expression in freshly differentiated adipocytes, depending on IFNγ. Altogether, our results suggest that PD-L1 is overexpressed in the WAT of obese individuals during IFNγ secretion, leading to T cell dysfunction and notably reduced cytolytic activity. Such a mechanism could shed light on why adipose-tissue-infiltrating viruses, such as SARS-CoV-2, can worsen disease in obese individuals.  Effective responses to intracellular pathogens or tumors are usually dependent on Natural killer (NK) cells and/or T cells, especially CD8+ cytotoxic T cells. NK cell killing can be regulated by killer cell inhibitory receptors (KIR) after being bound to HLA class I molecules . Exhaust2 is an independent risk factor for severe forms of COVID-19, as demonstrated in several clinical departments including ours, where 25% of hospitalized patients and 35% of ICU patients below the age of 70 were found to be obese [Obesity with a BMI of >30 Kg/mbe obese ,14. Indebe obese . Besidesbe obese . Indeed,be obese ,18. Moren = 5–6/group). One group was fed a standard diet  and another was fed a high-fat/high-sucrose diet . Food was given three times a week. Body weight was recorded weekly.All procedures were performed with the approval of the Regional Committee of Ethics for Animal Experiments (CECCAP), registered under number C2EA15 by the French Ministry of Higher Education and Research, in accordance with European directive 2010/63. The experimental protocol received the number 2018061411298503. Four-week-old C57Bl/6J male mice  were randomly housed, two per cage, at 21 °C with a normal light/dark cycle, free access to water, and a standard chow diet  for a one-week acclimatization. Next, mice were randomly assigned to four different groups , plasma levels of insulin , leptin, and adiponectin  were measured.2), or visceral surgery from lean controls with the approval of the Committee for the Protection of Human Subjects of the “Hospices Civils de Lyon” number 12/111 and with the patient’s consent, according to ethical principles for medical research involving human subjects, as described in the World Medical Association’s Helsinki Declaration. AT samples (50–100 mg) were mechanically dissected and incubated in 2 g/L of collagenase type Ia solution  in DMEM:F12 medium (50/50 vol/vol)  for 40 min at 37 °C by mixing. Isolation of ASC was performed as previously reported [Subcutaneous or visceral AT samples were isolated from residues of bariatric surgery from obese subjects  were obtained from healthy human blood donors. MNC were separated from blood through Ficoll-Histopaque density gradient centrifugation (Sigma-Aldrich) and stored in liquid nitrogen.ASC were harvested and seeded in 24-well plates  for 24 h in RPMI supplemented with 10% FCS. Then, 24 h later, MNC were added in the presence or absence of phytohemagglutinin . ASC/MNC ratios of 1:5 were used. For mRNA analyses, the culture period was 24 h, whereas it was 48 h when conditioning media were collected. In that latter case, IL-17A neutralizing antibodies  at 50 μg/mL or IFNγ-neutralizing antibodies  at 50 μg/mL were eventually added from the beginning of cultures. For blocking experiments, ASC/MNC cocultures were treated with PD-L1 neutralizing antibodies  at 1 or 10 μg/mL, or with irrelevant polyclonal goat IgG  at 10 μg/mL, or Secukinumab at 50 μg/mL.Total RNA was extracted from cocultures using the Tri Isolation ReagentTM  and frozen at –80 °C, and RT-qPCR was performed from reversely transcribed RNA into cDNA, as previously described . mRNA waFluorescein isothiocyanate (FITC)–, phycoerythrin (PE)- Allophycocyanin (APC)-, and Alexa-fluor- directly conjugated anti-human antibodies were used to stain the various cells tested: CD14 , CD73 , CD8 , CD274 , CD279 , and Granzyme B . Analyses were performed using a LSR II 3 lasers cytofluorometer and Diva software . For Granyme B intracellular staining, cells were permeabilized using the BD Cytofix/Cytoperm permeabilizing kit , and cells were analyzed 6 h after interaction with adipocytes. Ob-ASC were differentiated into AD as previously described .ATs were fixed in formaldehyde 3.7% after isolation from bariatric surgery. They were then embedded in paraffin before being cut with the microtome MICROT002 and mounted onto microscope slides for analysis, after staining with anti-human PD-L1  at 1:50 dilution.p was <0.05.Statistical analyses were performed using one-way ANOVA with or without repeated measures, or mixed-effects analyses , followed by post hoc Fisher’s LSD tests. In some experiments, two-tailed unpaired t-tests were used. Prism software was employed for analysis . Differences were considered as statistically significant when Whereas blood T cells from obese individuals or mice have been shown to overexpress the PD-1 checkpoint , here wePDL1 mRNA expression levels, but the co-presence of ASC resulted in potentiating the expression of PDL1 mRNA, whether ASC were issued from lean or obese donors. However, ob-ASC upregulated PDL1 mRNA expression at a statistically significant higher level than lean ASC. In addition, a two-way ANOVA test demonstrated (i) that both the body mass index (BMI) and the activation status were statistically significant independent factors, (ii) as well as the interaction between these two factors  from obese individuals (ob-ASC), rather than from lean individuals, can promote inflammation through Th17 cell and monocyte cell activation , we then factors A. Then, Because PD-1 needs to be linked by PD-L1 to exert its inhibitory effect on T cells, we then investigated whether the presence of obese ASC could also upregulate its expression. As shown in IL1Β and IL6 mRNA expression levels were not modified, thus demonstrating a specific PD-L1 inhibitory effect on T cell functions. The specificity of the anti-PD-L1 effects was supported by further experiments comparing IL-17 and PD-L1 blockade  collected from ob-ASC and MNC cocultures could propagate exhaustion. Therefore, we cultured freshly differentiated adipocytes from ob-ASC with CM harvested from PHA-activated cocultures. We observed a strong upregulation of the expression of re model , we thenpression A. To valpression B.Brown adipocytes and differentiated 3T3 cell lines have been shown to express PD-L1 ,28. AddiIL2 mRNA expression (IL-2 is a cytokine that plays a major role in the maturation of precytotoxic T cells into cytotoxic T cells (CTL) in order to allow them to subsequently exert their cytotoxic function . We havepression . Of intepression , thus suPD-1 was also found to be upregulated following activation of MNC with PHA . HoweverAltogether, these data lead us to suggest that increased expression of PD-L1 on adipocytes and of PD-1 on T cells may trigger exhaustion and inhibit T cell functions, in particular IL-2 secretion and subsequent CTL function. This could be of high importance in situations where viruses infiltrate WAT. Infection with SARS-CoV-2 could represent one such situation, as AT is known to express ACE2, a receptor for the spike protein of SARS-CoV-2 . InteresThis study was performed first in vivo in animals and then in in vitro coculture assays with human ASC and MNC. These assays have been used to investigate the mechanisms leading to increased expression of immune checkpoints, as observed in vivo. Although these coculture assays have been previously used to demonstrate the role of ASC, Th17 cells, and monocytes in initiating inflammation among AT from obese individuals , they haIn this study, we analyzed the impact of obesity on immune checkpoint expression in WAT, using a mouse model of diet-induced obesity. We observed that PD-L1 is overexpressed in WAT of obese mice, and is associated with increased expression of PD-1 in visceral WAT as compared with lean mice. Using human in vitro cocultures with ASC from obese individuals and MNC, we also observed an overexpression of PD-L1 with respect to cocultures with ASC from nonobese individuals, Moreover, conditioned media harvested from these cocultures enhanced PD-L1 expression in freshly differentiated adipocytes, depending on the presence of IFNγ. As adipocytes differentiated from obese ASC impaired cytotoxic activity, our results suggest that PD-L1 overexpression may occur in the visceral WAT of obese individuals under IFNγ secretion and may lead to T cell dysfunction, notably decreased cytolytic activity. Such a mechanism could shed some light on why adipose tissue-infiltrating viruses such as SARS-CoV-2 can worsen disease in obese individuals.


To better understand the impact of ICP22 binding to CD80 on HSV-1 infectivity and pathogenicity, we mapped the region of ICP22 required to bind the CD80 promoter to a 40-amino-acid (aa) region of ICP22. We constructed a recombinant HSV-1 expressing a truncated form of ICP22 that lacks these 40 aa, which does not bind to the CD80 promoter (KOS-ICP22Δ40) and retains the ability to replicate efficiently in rabbit skin cells, in contrast to ICP22-null virus. The replication of this recombinant virus in vitro and in vivo was higher than that of the ICP22-null virus, but virus replication kinetics were lower than those of the wild-type (WT) control virus. Similar to ICP22-null virus, the KOS-ICP22Δ40 mutant virus increased CD80 expression in dendritic cells (DCs) and interferon gamma (IFN-γ) expression in CD8+ T cells but not CD4+ T cells in infected mouse corneas. In contrast to the significantly reduced virus replication in the eyes of ocularly infected mice, the levels of latency reactivation were similar between KOS-ICP22Δ40 virus and WT virus. Thus, blocking ICP22 binding to the CD80 promoter using a recombinant virus expressing a truncated ICP22 that lacks CD80 promoter binding appears to reduce virus replication and enhance CD8+IFN-γ+ infiltrates in corneas of infected mice, with no effect on latency reactivation.Previously, we reported that herpes simplex virus type 1 (HSV-1) ICP22 binds to the CD80 promoter and suppresses its expression IMPORTANCE Direct binding of HSV-1 ICP22 to the CD80 promoter downregulates the expression of the costimulatory molecule CD80 but not CD86. In this study, we fine mapped the region of ICP22 required for binding to the CD80 promoter and constructed a recombinant virus containing a deletion in ICP22 that failed to bind to the CD80 promoter. This recombinant virus replicated less efficiently in vitro and in vivo than did the WT control virus, although CD80-expressing CD11c+ cells and IFN-γ-expressing CD8+ T cells were increased. Interestingly, the levels of latency and reactivation in the two viruses were similar despite lower virus replication in the eyes of infected mice. Therefore, blocking the interaction of ICP22 with the CD80 promoter could be used to temper the immune response. Followce used 813. Full  signals .ICP22 gene failed to suppress CD80 expression despite significantly reducing virus replication both in vitro and in vivo, leading to enhanced eye disease  HSV-1 suppresses the expression of the costimulatory molecule CD80, but not CD86, in corneas of ocularly infected mice . A varie16–22– disease . TherefoICP22 gene, but not any of the other HSV-1 genes, represses CD80 expression by removing the ability of the ICP22 protein to directly bind to the CD80 promoter   function of CD8, thus blocking cell lysis.HSV-1 carries at least 85 genes , and HSVpromoter . The abiing (CS) . We haveing (CS) . Becausein vitro and in vivo. We now have defined the amino acids required for ICP22 binding to the CD80 promoter and have deleted these amino acids in a recombinant virus . Our results show that (i) in contrast to the parental virus and similar to WT KOS, KOS-ICP22Δ40 virus grew in rabbit skin (RS) cells; (ii) in contrast to WT KOS, KOS-ICP22Δ40 virus expresses a truncated form of ICP22; (iii) in contrast to WT KOS, KOS-ICP22Δ40 virus did not suppress the expression of CD80 on dendritic cells in the corneas of infected mice; (iv) KOS-ICP22Δ40 virus replication is intermediate to those of ICP22-null and WT viruses both in vitro and in the eyes of ocularly infected mice; (v) levels of CD8+ interferon gamma-positive (IFN-γ+), but not CD4+IFN-γ+, cells increased in the corneas of KOS-ICP22Δ40-infected mice compared with mice infected with the WT control virus; and (vi) latency reactivation of KOS-ICP22Δ40 and WT KOS was similar for both viruses.Since the complete deletion of ICP22 increases CD80 and CD8 expression in corneas of infected mice, leading to increased eye disease , the curin vitro and in vivo (ICP22 gene construct with two in-frame copies of a FLAG tag at the 3′ end (We have demonstrated that ICP22 binds to the CD80 promoter and that this binding suppresses CD80 expression  in vivo . Our pree 3′ end  to assese 3′ end . To extee 3′ end . These fe 3′ end .pGL4-EV (control plasmid) or pGL4-CD80p DNA was transfected into HEK 293 (293) cells. The cells were also cotransfected with plasmids expressing ICP22 fragment A, B, or C, and their effect on CD80 promoter activity was determined by measuring luciferase activity 48 h later as we described previously . The effTo further map the suppressive region of ICP22 within fragment C, we created three new ICP22 fragments by dividing fragment C into three fragments: D  (40 aa),P < 0.05). Thus, the deletion of aa 290 to 369 eliminated the suppression of the CD80 promoter by ICP22 and is consistent with the suppressive region of ICP22 being within aa 290 to 369. To further map this activity based on fragment G and overlapping fragments E and F (P < 0.0001). These results suggest that the region of ICP22 that suppresses CD80 promoter activity is located within ICP22 aa 305 to 345.Based on fragments E and F, we created an ICP22 construct lacking 79 aa of ICP22 from aa 290 to aa 369 having an ATG, a 2× FLAG tag, and a termination codon . In cont E and F , we crea E and F . This co E and F , ICP22-Hin vitro and in vivo. D22 recombinant virus lacking the entire ICP22 gene was derived from the parental HSV-1 KOS strain (in vitro and in vivo (ICP22 gene with a deletion of 40 aa (aa 305 to 345) within ICP22 (pICP22Δ40). This plasmid also contained 463 bp of KOS sequence upstream of the ICP22 ATG and 554 bp of KOS sequence downstream of the ICP22 termination codon. We used D22 as the parental virus to generate KOS-ICP22Δ40 recombinant virus by cotransfecting pICP22Δ40 DNA and D22 DNA into RS cells as we described previously . Human embryonic kidney (HEK) 293 cells were infected with HSV-1 WT KOS, D22 having a complete deletion of ICP22, or KOS-ICP22Δ40 having a recombinant ICP22 with a 40-aa deletion or were mock infected. As expected, neither mock nor D22 expressed a detectable ICP22 product , 36, 413P > 0.05) (P < 0.001) (P < 0.05) (P < 0.001)  ( > 0.05) , but by < 0.001) . At 1 PF < 0.05) , 24 h an< 0.001) . Similarin vivo, BALB/c mice were infected ocularly with 2 × 105 PFU/eye of the KOS-ICP22Δ40 mutant virus, WT KOS, or D22. Previously, we showed that D22 replicated at significantly lower levels in mouse eyes than WT KOS (P < 0.01) (P < 0.001)  . Similar< 0.001) . These red above  and may 5 PFU/eye of KOS-ICP22Δ40 or KOS virus. Individual TG from 18 to 20 infected mice were harvested on day 28 p.i., and latency levels were determined by real-time PCR or quantitative reverse transcription-PCR (qRT-PCR) based on HSV-1 latency-associated transcript (LAT) expression as described in Materials and Methods. Unlike the lower viral replication in the eyes of KOS-ICP22Δ40-infected than in WT KOS-infected mice (P > 0.05)  . This is > 0.05) .5 PFU/eye of HSV-1 KOS-ICP22Δ40 and KOS after corneal scarification. Virus reactivation was analyzed by explanting individual TG from 10 infected mice on day 28 p.i. as described in Materials and Methods. As with latency levels, mice infected with KOS-ICP22Δ40 or KOS virus had similar reactivation times (P > 0.05)  . Howeverin vitro and in vivo and that a recombinant HSV-1 lacking ICP22 upregulated CD80 and IFN-γ expression in the corneas of ocularly infected mice  analysis revealed that CD80 expression in DCs was higher in KOS-ICP22Δ40-infected mice than in KOS-infected mice (2.19% versus 0.99%) (+CD80+ expression in the corneas of infected mice than in mice infected with WT KOS virus (P < 0.05)  (+IFN-γ+ cells in the corneas of infected mice than in those from WT KOS virus-infected mice (P < 0.05)  (P > 0.05)  . The exp < 0.05) . The pers 2.67%) , and res < 0.05) . The pers 0.18%)  and were > 0.05) . Thus, aing CD80 , 38.in vitro and in vivo, and (iii) compare the immunogenicities of this recombinant virus and the WT virus relative to primary HSV-1 infection, immune infiltrates in the eye, and latency reactivation in ocularly infected mice.Our previous studies demonstrated that (i) HSV-1 ICP22 downregulates the expression of CD80 but not that of CD86, in the presence or absence of anti-HSV-1 antibodies ; (ii) a in vitro and in vivo. ICP22 is one of the five HSV-1 immediate early genes, and the initial steps of replication begin with these five ICP proteins  play any role in CD80 suppression.In this study, we demonstrated that KOS-ICP22Δ40 virus significantly enhanced the expression of CD80 by CD11c in the corneas of ocularly infected mice relative to WT virus. These results are consistent with our previous study showing that infection of mice with WT virus suppressed CD80 expression in DCs , while t disease , 38. Furscarring . These rin vitro and in vivo, mice infected with this virus had levels of latency and reactivation similar to those of the WT virus. This is likely a consequence of immune escape due to a lack of ICP22 binding to the CD80 promoter. The lack of a correlation between viral load, latency, and reactivation is probably due to higher CD80 expression levels. However, the positive correlation between latency and reactivation is consistent with our previous studies  were used.Triply plaque-purified WT KOS , D22 , and KOS-ICP22Δ40 viruses were used in this study and grown as we described previously . The D22eviously , 33. Rabeviously . Cells wICP22 gene with a 40-aa deletion corresponding to aa 305 to 345 of ICP22. This construct also contained 463 bp of KOS sequences upstream of the ICP22 ATG and 554 bp downstream of the ICP22 termination codon. The resulting plasmid, designated pICP22Δ40, contains 1,152 bp of the truncated ICP22 gene bounded by 463- and 554-bp adjacent regions of ICP22 sequences.The truncated ICP22 plasmid was constructed using sequences of HSV-1 strain KOS . We syntICP22 gene was completely deleted. D22 virus grows in Vero cells but not in RS cells (ICP22 gene by PCR using ICP22 forward (GCGCCACCTGATACGCGACTG) and reverse (CGACCGCAGACAGCCAGGGC) primers. The PCR product length for WT ICP22 was 458 bp, and that for truncated ICP22 was 338 bp. All selected plaques were verified as having 338-bp amplified products, and a single plaque chosen for purification was designated KOS-ICP22Δ40.The parental virus for the KOS-ICP22Δ40 construct was D22, a mutant of the HSV-1 KOS strain in which the RS cells . KOS-ICPRS cells . BrieflyICP22 gene  and two copies of FLAG sequences inserted into the BamHI site of pcDNA3.1. We also synthesized ICP22 fragments corresponding to different regions of ICP22 . Using a dual-luciferase reporter system , two individual reporters were introduced simultaneously to determine responses within the same cells. Sample preparation was conducted as described by the manufacturer (Promega). Cells were washed with phosphate-buffered saline (PBS) and lysed in lysis buffer, and the collected supernatants were transferred to 96-well plates. The luminometer  was primed with luciferase and Stop & Glow reagents. Assays were conducted in replicates of 10, and means ± standard errors of the means (SEM) were calculated from three separate experiments (n = 30).Transfection experiments were conducted using 293 cells and Gene Porter 2  as we described previously . BrieflyRS cell monolayers at 70 to 80% confluence were infected with 0.1 or 1 PFU/cell of WT KOS or KOS-ICP22Δ40. Virus was harvested at 24 and 48 h postinfection by two cycles of freeze-thawing of the cell monolayers with medium. Virus titers were determined by standard plaque assays on Vero and RS cells.5 PFU/eye of HSV-1 strain KOS, KOS-ICP22Δ40, or D22 with corneal scarification as we described previously . The amplicon length for the LAT primer set is 81 bp, corresponding to LAT nucleotides (nt) 119553 to 119634. Relative LAT RNA copy numbers were calculated using standard curves generated from the pGem5317 plasmid as we described previously  buffer, and centrifuged in microcentrifuge tubes at 14,000 rpm for 15 min. The supernatant of each lysate was transferred to a new tube and kept on ice. Lysate protein concentrations were measured using a reducing-agent-compatible bicinchoninic acid (BCA) protein assay kit . We loaded 40 μg of the total whole-cell lysate onto each lane of a 10% SDS-PAGE gel for electrophoresis. The blot was probed overnight using primary anti-ICP22 at 1:5,000, rabbit polyclonal antibody 413 , and β-actin monoclonal rabbit antibody at 1:5,000 . For detection, a secondary anti-rabbit IgG horseradish peroxidase (HRP)-linked antibody was diluted at 1:4,000  and incubated for 2 h. Polyvinylidene difluoride (PVDF) membranes  were washed three times with 1× Tris-buffered saline–Tween (TBS-T) and developed using Signalfire ECL .5 PFU/eye of KOS-ICP22Δ40 or WT KOS virus. Corneas from three mice were combined, and single-cell corneal suspensions were prepared as we described previously  using GraphPad v9 . Results were considered statistically significant if the P value was <0.05.Data were analyzed by Student’s


To evaluate objective criteria of Magnetic Resonance Imaging (MRI) of Placenta Accreta Spectrum disorder (PAS) analyzing interobserver agreement and to derive a model including imaging and clinical variables to predict PAS.A retrospective review including patients submitted to MRI with suspicious findings of PAS on ultrasound. Exclusion criteria were lack of pathology or surgical information and missing or poor-quality MRI. Two radiologists analyzed six MRI features, and significant clinical data were also recorded. PAS confirmed on pathology or during intraoperative findings were considered positive for the primary outcome. Variables were tested through logistic regression models.Final study included 96 patients with a mean age of 33 years and 73.0% of previous C-sections. All MRI features were significantly associated with PAS for both readers. After logistic regression fit, including MRI signs with a moderate or higher interobserver agreement, intraplacental T2 dark band was the most significant radiologic criteria, and ROC analysis resulted in an AUC = 0.782. After including the most relevant clinical data (previous C-section) to the model, the ROC analysis improved to an AUC = 0.893.Simplified objective criteria on MRI, including intraplacental T2 dark band associated with clinical information of previous C-sections, had the highest accuracy and was used for a predictive model of PAS. The Placenta Accreta Spectrum disorder (PAS) encompasses previous terms such as morbidly adherent placenta, placental invasion, and abnormally invasive place,The most common risk factors for PAS are prior cesarean delivery and placenta previa, with rising incidence in the past years.,3 MoreovUltrasound is the most widely used method for the diagnosis during the prenatal period given wide availability and relatively low-cost method and has a good accuracy mainly for the anterior uterine wall evaluation. Besides that, ultrasound allows a longitudinal assessment of women at risk for PAS increasing its possibility to reach the final diagnosis.[17]Several studies have evaluated MRI criteria associated with PAS; however, most articles evaluated isolated criteria, had smaller sample sizes, or did not have pathological and surgical confirmation., 24, 25 The Institutional Review Board (IRB) approved this retrospective study and granted a waiver of consent. This is a tertiary hospital, and the obstetric department is a reference for high-risk patients. Patients that are diagnosticated by ultrasound with placenta previa and have the suspicion of PAS are routinely sent to MRI. Therefore, the authors included in the study pregnant women who performed aj MRI under those circumstances during the period from July 2008 to December 2017. At last, the authors had a total of 110 patients, but 14 patients were not included in the analysis for different reasons, such as an anatomopathological study of the placenta not available (n = 2), surgical description not available (n = 3), MRI images not available (n = 2) and low-quality MRI images (n = 7) .Fig. 1FlThe following clinical data were also recorded: maternal age, gestational age at MRI, gestational age at delivery, the time between MRI and delivery, ethnic group, parity, previous C-sections, abortions, history of curettage, and other uterine surgeries. PAS confirmed on the pathology of surgical specimen and/or signs of PAS during intraoperative findings were considered as positive for the primary outcome .Table 1CAll MRI examinations were performed on a 1.5T unit  with body array coils, including axial, coronal, and sagittal T2-weighted Single Shot Fast Spin-Echo (SS-FSE); axial, coronal, and sagittal balanced steady-state free precession (Fast Imaging Employing Steady-State Acquisition – FIESTA); axial and sagittal fat-suppressed ultra-fast spoiled gradient-echo T1-weighted (LAVA) without intravenous contrast media.The parameters for SS-FSE images were Repetition Time (TR)/Echo Time (TE), 650–16000 / 50–90 msec; Flip Angles (FA), 90; Field of View (FOV), 120–480 mm; slice thickness, 4.5 mm. For balanced ‒ steady-state free-precession ‒ FIESTA images, they were TR/TE, 3800–6000/ 50–90 msec; FA = 90; FOV, 120–480 mm; slice thickness, 4.5 mm, and for T1 LAVA images, they were: TR/TE, 200–230 / 1.8‒11 msec; FA = 80; FOV, 340–380 mm; slice thickness, 4.5 mm.Two board-certified blinded radiologists with different levels of expertise  retrospectively analyzed the exams on a Likert scale1- Intraplacental abnormal vascularity: tortuous enlarged flow voids observed on T2 and corresponding high signal on FIESTA, indicating vascular flow - 5: > 6mm - 4: 5-6 mm - 3: 3-4 mm - 2: 1-2 mm - 1: absentIntraplacental T2 dark band: wedge-shaped areas of low signal intensity on T2-weighted images and FIESTA (19) , Fig. 4. - 5: > 20 mm - 4: 15-20 mm - 3: 10-14 mm - 2: < 10 mm - 1: absent2- Placental bulge: focal bulging of the uterine contour.,19,24,27 - 5: clear loss of myometrial congruency or invasion of adjacent organs - 4: focal bulges - 3: disruption of the normal pear shape of the uterus with the lower uterine segment being wider than the fundus - 2: slight irregularities on uterine contours - 1: normal contours3- Placental protrusion sign: placenta pushing inferior to the internal os.[21] - 5: > 15 mm - 4: 11-15 mm - 3: 6-10 mm - 2: < 5 mm - 1: absent4- Myometrial thinning: focal thinning and indistinctness of the myometrium and loss of thin dark uteroplacental interface on T2-weighted images.,28,295- Heterogeneous placenta: marked heterogeneous intensity within the placenta .18,30, , 32Fig. The MRI signs used for evaluation were:The surgical description had three outcomes: normal discharge of the placenta, focal attachment , and hysterectomy. Only hysterectomy was considered positive for PAS. For pathological analyses, there were five outcomes: normal placenta, focal acretism, accreta, increta and percreta. Normal placenta and focal acretism were considered negative for pathological PAS.t-Student test for parametrical variables.Descriptive analyses of the data were performed by using absolute and relative frequencies, central tendency, and dispersion measures. The comparison between the groups of PAS and no PAS was made with a Mann-Whitney test for non-parametrical variables and The interobserver variability of all MRI signs was assessed using the Kappa (κ) value. A κ-value of ≤ 0.20 was interpreted as slight agreement, 0.21–0.40 fair agreement, 0.41–0.60 as moderate agreement, 0.61–0.80 as substantial agreement, and ≥ 0.81 as almost perfect agreement.[33]For the development of the predictor model, both readers reassessed the discordant criteria and reviewed it in consensus. This strategy was made to extract the best possible information from the MRI and build the best predictor model possible. To identify which MRI signs and clinical variables were most significant, analyses through multiple binary logistic regression models were made in other to identify its coefficients, Odds Ratios (OR), and their respective 95% Confidence Intervals (95% CI). The significant variables (p < 0.005) were tested on the multiple modeling and those which presented a value of p < 0.20 by the Stepwise technique.Receiver Operating Characteristic (ROC) analysis was used for evaluation of the diagnostic performance using only the most significant MRI sign and using the most significant MRI sign combined with the most significant clinical data for PAS. The areas under the ROC Curves (AUC) were estimated nonparametrically for non-ordinal score assessments.Analyses were conducted using statistical software .The final sample of 96 patients was divided into two groups according to anatomopathological study and/or surgical descriptions: patients with PAS (n=42) and patients without PAS (n=54) .For all the clinical variables analyzed, only parity and previous C-sections showed significant differences between the PAS and no PAS groups. All other variables: abortions, curettage, maternal age, gestational age at MRI, gestational age at delivery, and time interval between MRI and delivery showed no significant differences between the PAS and no PAS groups .Table 2AAll MRI signs  showed significant differences between the PAS and no PAS groups for both reader A and reader B .Table 3MThe Sensitivity (SEN), Specificity (SPE), Positive Predictive Value (PPV), and Negative Predictive Value (NPV) for each MRI sign are shown in .Concordance coefficient values are shown in . IntraplInitially, in other to create a reproducible predictor model, the authors excluded the MRI signs that showed a slight agreement  and fair agreement  in the interobserver analysis.The remaining MRI signs  and the most relevant clinical sign (previous C-section) were analyzed using a multiple binary logistic regression model. The most significant clinical variable in isolation was the presence of a previous C-section with an OR = 3.35 (95% CI 1.88‒5.97), and the most significant MRI sign was intraplacental T2 dark band with an OR = 12.67 (95% CI 3.97‒40.45) as shown in The result of ROC analyses for the prediction of PAS using only the most significant MRI sign  and using the most significant MRI sign combined with the most significant clinical data (previous C-sections) are shown in Finally, the authors derived a predictor model for the estimated PAS probability according to the number of previous C-sections for patients that had or not the presence of the intraplacental T2 dark band sign .The analysis in the present study has confirmed that MRI signs of PAS  were associated with the presence of PAS for both inexperienced and experienced radiologists as observed in previously published studies.The authors have also evaluated the interobserver variability of MRI analyses of experienced and less experienced radiologists, knowing that the previous experience of the radiologist improves the diagnostic performance, as shown by Ghezzi et al.[34]Regarding clinical variables, they can be important risk factors for the development of PAS, mainly in relation to the previous C-section, as previously shown.The predictive model, including only MRI sing , had great accuracy (AUC = 0.782), which was further improved (AUC = 0.893) when adding previous C-sections. These results demonstrate that this model can be useful for obstetricians to evaluate the estimated probability of PAS on delivery very easily so that they can properly manage it in advance and are not surprised with a potential high-risk procedure.There are some limitations of the present study. To begin with, it was performed in a tertiary hospital with high-risk patients selected from abnormal ultrasound, inducing selection bias. Moreover, radiologists in the study were well trained for this rare condition, given the particularity of the hospital, and may not reflect standard radiology practice. Some of the advantages of the study are its high number of patients included compared with previous studies and the use of a gold standard combining surgical description and pathological analyses. According to the sample, the results must be analyzed carefully. The reduced sample size contributes to reproducibility issues, including false positives and false negatives. However, the model was analyzed considering the number of outcomes to avoid an overfitting model.Simplified objective criteria on MRI  combined with clinical data (previous C-sections) contributed to the creation of a predictive model, which the authors believe can facilitate and improve the diagnostic accuracy by providing a more objective result for the MRI report. Besides, it allows a higher uniformization of the analyses between radiologists with different levels of expertise and facilitates information to obstetricians. Based on these findings, the authors suggest the application of this model in prospective studies in order to elucidate external validation issues.Rodrigo Pamplona Polizio: Conceptualization, methodology, data collecting, writing-original draft.Fernando Ide Yamauchi: Conceptualization, methodology, data collecting, review the final version.Renata Franco Pimentel Mendes: Data collecting, review the final version.Stela Verzinhasse Peres: Formal analysis.Mario Macoto Kondo: Review the final version.Rossana Pulcineli Vieira Francisco: Conceptualization, metodology, writing-review & editing.The authors declare no conflicts of interest.


These test kits and on-mobile DIC were used for the on-site determination of nutrients in the Pak Bang and Bang Yai canals, the main canals in Phuket, Thailand. The concentrations of nitrite, nitrate, and phosphate were undetectable to 0.60 mg L−1, undetectable to 2.98 mg L−1, and undetectable to 0.52 mg L−1, respectively.Portable colorimetric hydrogel test kits are newly developed for the on-site detection of nitrite, nitrate, and phosphate in water. Griess-doped hydrogel was prepared at the bottom of a 1.5 mL plastic tube for nitrite detection, a nitrate reduction film based on zinc powder was placed on the inner lid of a second 1.5 mL plastic tube for use in conjunction with the Griess-doped hydrogel for nitrate detection, and a molybdenum blue-based reagent was entrapped within a poly hydrogel matrix placed at the bottom of a third 1.5 mL plastic tube to detect phosphate. These test kits are usable with on-mobile digital image colorimetry (DIC) for the on-site determination of nutrients with good analytical performance. The detection limits were 0.02, 0.04, and 0.14 mg L Water pollution is a major challenge that humanity is facing. Specifically, nutrient pollution caused by excess nitrogen and phosphorus in the water can have diverse, extensive effects on public health, environments, and economies. Nitrogen and phosphorus are the most important and abundant nutrients that occur naturally in aquatic ecosystems. However, substances from human activities, such as fertilizers, wastewater, automobile exhaust, and animal waste, introduce excess amounts of these nutrients into the ecosystem faster than it can adapt. This results in their overabundance, leading to increased primary production of biomass, oxygen depletion, and toxic algal blooms (eutrophication) . Eutroph2−) and nitrate (NO3−) are nitrogen nutrients that are integral parts of the nitrogen cycle in the environment, whereas phosphates are the most common phosphorus nutrients. Nitrite is an important indicator of water quality, and it is generally found in surface water at low concentrations (0.07 mg L−1 NO2−–N) because it oxidizes to nitrate easily. Orthophosphates are the most stable phosphates, and they can exist in a variety of species, such as orthophosphate/phosphate (PO43−), hydrogen phosphate (HPO42−), dihydrogen phosphate (H2PO4−), and phosphoric acid (H3PO4), based on different pH values [−1 to 0.03 mg L−1 [−1 [−1, respectively [Nitrite  Griess and molybdenum blue-based reagents were entrapped within a poly (PVA) matrix to fabricate small, portable test kits in 1.5 mL plastic tubes for nitrite and phosphate, respectively. For nitrate detection, zinc powder was entrapped within a tapioca starch film to fabricate a nitrate reduction film on the inner lid of the 1.5 mL plastic tube containing the Griess-doped PVA nitrite test kit. This film reduces nitrate to nitrite, which is then subjected to a colorimetric reaction with the Griess-doped PVA nitrite test kit at the bottom of the tube. These test kits will make the colorimetric testing of these nutrients easier and more portable while eliminating any risk from hazardous chemicals. They are also environmentally friendly methods since PVA and tapioca starch are biodegradable polymers. Digital image colorimetry (DIC) based on a smartphone application (on-mobile DIC) was used in conjunction with the developed hydrogel test kits to eliminate the need for a spectrophotometer ,9. This The in-tube hydrogel test kits for nitrite a and nitw/v ammonium molybdate, 10 mL 4.5 M sulfuric acid, and 1 mL 1.25% w/v potassium antimony tartrate) and the 20% ascorbic solution was determined. The findings showed that 3:1 provided the darkest color product with the lowest RGB intensities  was also optimized from 5% to 15%, and a 10% w/v PVA solution provided the darkest color product  was then optimized, and results indicated that 0.25 mL provided the darkest color product with good characteristics (w/v PVA solution was mixed with 0.25 mL molybdenum blue-based reagent to prepare the polymer mixture for phosphate detection. The amount of the cross-linker (EGDE) was also optimized by adding it (1 to 10 µL) into the obtained polymer mixture (1.25 mL), and 2.5 µL was selected as the optimal value because it yielded the darkest color product and good hydrogel characteristics  and chemical cross-linking (use of EGDE as a cross-linker), as done in our previous studies ,19 but m−1 was attributed to the O–H stretching from sulfuric acid and/or ascorbic acid; this may have overlapped with the O–H stretching from the PVA, which appeared as a broad absorption peak at 3268 cm−1 in the blank hydrogel (PVA and EGDE without reagent) [−1 [−1, a slight shift from that of the blank hydrogel  [−1 [−1. The peak at 1676 cm−1 was assigned to the C=C stretching from ascorbic acid [−1 might be attributed to the vibration of enol hydroxy [−1 might be attributed to the Sb–O from potassium antimony tartrate [The characterization of the hydrogel test kit for phosphate was investigated. The scanning electron microscopy (SEM) images of the test kit revealed a homogeneous entrapment of the molybdenum blue-based reagent within the hydrogel matrix a,b. The reagent) . It may ent) [−1 . A stret910 cm−1 ). The st910 cm−1  was obse990 cm−1 , may havm−1 [−1)  and/or C[−1) [−1 ) and thubic acid ,24, whertartrate .Pink-violet products were obtained from nitrite and nitrate testing, whereas blue products were obtained from phosphate testing . They weR, IG, and IB) of the blue products (2.5 mg L−1) for −3.4%, −2.3%, and +0.9%, respectively. Moreover, the hydrogel test kit for nitrite can be frozen (−20 °C) for three months, and the nitrate reduction films can be stored in a desiccator (25 °C) for six months [The blue products darkened with increasing reaction time a, and 10x months . Therefo−1) to pH 1 to 12 using HCl and NaOH before testing. The results  were obtained from the analysis of the color products  and red (IR), which agrees with a previous report [B was higher than IG and IR because of the higher reflection of blue light  and limit of quantification (LOQ) (0.07 mg L−1) for nitrite analysis were lower than those of nitrate . These are lower than the concentration limits in surface water recommended by the WHO of 3 and 50 mg L−1 for nitrite and nitrate, respectively [−1, equivalent to 0.05 and 0.15 mg PO4–P L−1, respectively. This LOD is lower than the maximum concentration of total phosphorus in discharge from municipal wastewater treatment plants in Thailand (2 mg L−1 [4–P in flowing water by the US EPA (≤0.1 mg L−1) [−1, [4–P in streams at points where they discharge into lakes or reservoirs (≤0.05 mg L−1) [−1) against the concentration quantified by the IR calibration graph was +3.0%, whereas +3.0% and −4.8% RE were obtained from the IG calibration graphs for nitrite and nitrate, respectively. These results indicate the excellent accuracy of the developed method. A good intraday precision (0.70% to 1.52% relative standard deviation (RSD)) was also obtained from testing 12 hydrogel test kits for phosphate, whereas the interday precision from nine test kits over three days was in the range of 0.70% to 1.69% RSD. The performance of the developed method was similar to that in other reports  ,38 and m mg L−1) ,38. The  reports , but it −1 were tested with the hydrogel test kits for phosphate. No color product was observed from any of the ions (−1) to investigate the tolerance ratios. The results in −1) using seawater sampled from the Andaman Sea (far from human activity) instead of ultrapure water. The sensitivity of the IR standard graph from seawater was different from that obtained using ultrapure water by −3.83%, indicating that the seawater matrix exerted little effect. Ascorbic acid and formic acid were also tested with nitrate, nitrite, and phosphate test kits, and no color products were observed. Phosphate (5 mg L−1) can tolerate ascorbic acid and formic acid at high concentrations  can tolerate formic acid at 10,000 mg L−1, but at lower concentrations for ascorbic acid (25 mg L−1).Various ions at ~10,000 mg Lthe ions , indicat−1, undetectable to 2.98 mg L−1, and undetectable to 0.52 mg L−1, respectively , whereas a mean phosphate concentration of 0.50 ± 0.03 mg L−1  was found by the developed method at sampling points P12 and P14. The higher mean concentration of the developed method was due to its LOQ of 0.47 mg L−1, which means that the method cannot quantify phosphate in the range of the standard method (0.30 to 0.42 mg L−1). The nutrient pollution index (NPI) was calculated by the sum of the nitrate, nitrite, and phosphate indices [iC is the mean concentration of nutrients in water and iMAC is the maximum allowable concentration of nutrients in surface water recommended by the WHO , moderate (1 ≤ NPI ≤ 3), considerable (3 < NPI ≤ 6), or very high (NPI > 6) [NPI of the Bang Yai canal (0.21) was lower than that of the Pak Bang canal (0.25), but both values indicated an absence of pollution, similar to the NPI of 0.27 at sampling points P10 to P15.The nitrate concentration was commonly higher than that of nitrite; this may have been due to the oxidation of nitrite to nitrate. It was also higher than that of phosphate, as in other reports ,46, and ectively . The higectively . Polluti−1, >99% hydrolyzed), zinc powder, and EGDE (98%) were from Sigma-Aldrich . Tapioca starch  was bought from a supermarket; a spectrophotometer showed that it had 27% amylose content, which accorded with the Thai agricultural standard TAS 4000-2003 . All standard solutions were prepared with ultrapure water from a water purification system .Sodium nitrate, sodium nitrite, ammonium heptamolybdate tetrahydrate, and potassium antimony tartrate were purchased from Carlo Erba . Disodium hydrogen phosphate and phosphoric acid (85%) were from Ajax Finechem , and sulfuric acid was obtained from J.T. Baker . NED and ascorbic acid were bought from Fisher Scientific , and sulfanilamide was purchased from Panreac . PVA  or molybdenum blue-based reagent (0.25 mL) was mixed with 1 mL of the PVA solution, the mixture was stirred for a few minutes, 2.5 μL EGDE was added, and the mixture was stirred for another minute. Each polymer mixture (100 μL) was transferred into the bottom of a 1.5 mL plastic tube and kept in a freezer for 24 h, and the result was the hydrogel test kits. Blank hydrogel test kits were also prepared using the same procedure but without the reagents.The hydrogel test kits for nitrite, nitrate, and phosphate detection were developed by entrapping Griess and molybdenum blue-based reagents within PVA hydrogel matrices. Each PVA solution was prepared by dissolving PVA granules (1 g) in ultrapure water (10 mL) at 120 °C and stirring the mixture for 30 min. The clear, viscous solution was cooled at room temperature before adding either the Griess or molybdenum blue-based reagent, and then a cross-linker (EGDE). The Griess reagent was prepared by dissolving 0.3 g sulfanilamide, 45 mg NED, and 1.5 mL 85% phosphoric acid in 10 mL ultrapure water. The molybdenum blue-based reagent was prepared by mixing 4 mL 10% The nitrate reduction film was prepared per our previous report . TapiocaThe morphology of the hydrogel test kit for phosphate was investigated using scanning electron microscopy , and the functional groups were investigated using Fourier-transform infrared (FTIR) spectrophotometry  with the KBr pellet method.−1) were prepared by diluting stock solutions (500 mg L−1) with ultrapure water. For colorimetric testing, the standard solutions (~1.5 mL) were added to the plastic tubes containing the hydrogel test kits and left for 10 min. Three replications were conducted at each standard concentration of nitrate, nitrite, and phosphate. DIC based on a custom-built smartphone application was then applied for quantitative analysis. The colorimetric product was placed in a sample holder inside a custom-built RGB analysis box (8.5 × 9.5 × 6.25 inches) modified from previous works [R, IG, and IB values of the color products against the standard concentrations.Standard solutions of sodium nitrate, sodium nitrite, and disodium hydrogen phosphate  against their established calibration curves and recovery percentage from real spiked samples.System performance parameters were investigated: the linear range, correlation coefficient, sensitivity, LOD, and LOQ. The LOD and LOQ were calculated based on the International Council for Harmonisation (ICH) harmonized tripartite guideline , whereasSurface water and seawater samples were randomly obtained from the Pak Bang canal, Bang Yai canal, and estuaries of both canals. A total of 135 grab samples were collected from 15 sampling points from the Pak Bang canal  of these colorimetric products can be analyzed using a custom-built smartphone application and used to quantify these nutrients with good analytical performance. The relationships between IG and the nitrite and nitrate concentrations were linear in the range of 0.1 to 1 mg L−1, with a good correlation coefficient (R > 0.99), similar to that of the relationship between IR and the phosphate concentration in the range of 0.25 to 5 mg L−1. The obtained LODs for these nutrients were lower than the maximum allowable concentration of nutrients in surface water recommended by the WHO; they were accurate (<4.8% RE) and precise (<1.85% RSD). These test kits and on-mobile DIC were then used for the on-site determination of nutrients in the Pak Bang and Bang Yai canals, which are the main canals in Phuket, Thailand. The nitrite, nitrate, and phosphate concentrations ranged from undetectable to 0.60 mg L−1, undetectable to 2.98 mg L−1, and undetectable to 0.52 mg L−1, respectively, which are under the limits set by the WHO standard. The NPIs of both canals indicated an absence of pollution.In-tube hydrogel test kits were used in conjunction with on-mobile DIC for on-site analysis of nutrients in surface water and seawater in Phuket, Thailand. Pink-violet products were obtained from nitrite testing using Griess-doped hydrogel test kits. A reduction film based on zinc powder was used to reduce nitrate to nitrite before testing the resultant nitrite with a Griess-doped hydrogel test kit for nitrate testing. Blue products were obtained from phosphate testing with a molybdenum blue-doped hydrogel test kit. The RGB intensities (I


CACNA1C gene–encoding for the major Ca2+ channel of the heart–may exhibit a variety of clinical manifestations. These include typical or atypical Timothy syndromes (TS) which are associated with multiple organ manifestations, and cardiac involvement in form of malignant arrhythmias, QTc prolongation, or AV block. “Cardiac only” Timothy syndrome (COTS) shows no extracardiac manifestation, whereas some CACNA1C gene mutations are associated with QTc prolongation alone .Mutations in the CACNA1C gene mutation associated syndromes, including TS, COTS and isolated LQT8 via major databases published from 2004 through 2019 was performed. Detailed patient-level phenotypic and genotypic characteristics, as well as long-term outcome measures were collected and compared between pre-specified patient groups, defined both on phenotype and genotype.A systematic search of the literature reporting cases of CACNA1C exon 8/8A vs. non-exon 8/8A mutation carriers. TS showed a high degree of genetic homogeneity, as the p.Gly406Arg mutation either in exon 8 or exon 8A alone was responsible for 70% of the cases.A total of 59 TS, 6 COTS, and 20 isolated LQT8 index cases were identified. Apart of syndactyly or baldness, there were no major differences regarding clinical manifestations or outcome measures between TS subtypes, either defining TS subtypes on the genotype or based on the phenotype. Both subtypes were characterized by an extreme degree of QTc prolongation (median ≥600 ms) which were reflected in high major adverse cardiac event rate. On the other hand, there were marked differences between TS, COTS, and isolated LQT8. Timothy syndrome was characterized by a much earlier disease onset, much more pronounced QTc prolongation and much higher mortality rate than COTS or isolated LQT8. Similar differences were observed comparing CACNA1C gene show important clinical differences. Timothy syndrome is associated with the most severe clinical phenotype and with the highest risk of morbidity and mortality. However, distinguishing TS subtypes, in any form, are not supported by our data.Clinical phenotypes associated with mutations in the https://www.crd.york.ac.uk/prospero/], identifier [CRD42020184737].[ The channel has several subunits, including the α1C-subunit, responsible for voltage sensing, for conduction pore formation, and for the gating mechanism. The fully functional channel complex is comprised of an additional intracellular β subunit and an extracellular α2δ subunit. The α1C subunit has 4 domains (I–IV), each with six transmembrane-spanning segments (S1–S6). S1–S4 are the voltage-sensitive subunits, whereas S5–S6 are the selectivity filter. When the membrane is depolarized, the S4 domain approaches the cytoplasmic ends of the S5 and S6 helices and the conduction pore opens. Changes in L-type calcium channel function  can affect cardiac myocyte contractility and arrhythmogenicity. The channel is expressed in many tissues of the body , which is characterized by QTc prolongation, AV-block, congenital heart defects, facial dysmorphisms, episodic hypoglycemia and neurologic symptoms including developmental delays, possible autism, seizures, and intellectual disability. The most distinctive morphological hallmark of TS1 is syndactyly, in contrast to atypical Timothy syndrome  patients, who have no syndactyly but carry many of the other multisystemic manifestations of the disease  (Carriers of exon 8A p.Gly406Arg 2)CACNA1C mutations [formerly categorized as Timothy syndrome subtype 2 (TS2) in the strictest sense] (Carriers of exon 8 p.Gly406Arg 3)CACNA1C mutations  (Carriers of exon 8 p.Gly406Arg and pGly402Ser  report] .4)CACNA1C mutations .Carriers of all exon 8 5)CACNA1C mutations.Carriers of non-exon 8/8A According to reported phenotypic manifestations patients were divided into the following three groups:1)TS . Two subgroups, i.e., typical TS (defined as patients with syndactyly) and atypical TS (defined as patients without syndactyly) were also analyzed separately.2)Cardiac only TS .3)Isolated long-QT syndrome 8 .Proven mosaic TS cases, reported as such, present in very small numbers in the reported publications, were not considered, taking the very specific genetic constellation of these cases into consideration.For all the comparator groups two sets of comparisons were made: (i) including index patients only  (ii) including all affected patients .CACNA1C mutations. All the mutations were re-evaluated in ClinVar1 and Varsome.2As many of the considered publications were published before the standards for the interpretation of sequence variants were issued by the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) in 2015,  we reassU-test or the Kruskal–Wallis test was used to detect differences between groups. A p < 0.05 value was considered as statistically significant. All calculation was made by IBM SPSS statistical software .For descriptive statistics the number of cases and percentage were calculated for each group in case of categorical and median and interquartile range in case of continuous variables. The differences between groups in case of. categorical variables were examined using the Chi-square test and Fisher’s exact test. For continuous outcomes, because of the origin of the data, non-parametric methods, such as Mann–Whitney Excluding reports on mosaic patients, –23 a totOut of the 134 patients, there were 85 index patients and 49 additional family members. Out of the 85 index patients 59 suffered from TS, 6 from COTS and 20 from isolated LQT8, respectively. In the entire patient population (index patients and relatives), there were 60 patients with TS, 15 patients with COTS and 59 patients with isolated LQT8 .CACNA1C mutations were extracted from the reports , 6 pts. with exon 8 p.Gly406Arg mutations, 12 pts. with exon 8 p.Gly406Arg/p.Gly402Ser mutations and 16 pts. with exon 8 mutations . In further five patients  it was not unequivocally reported and was impossible to determine whether the patients carried the p.Gly406Arg mutation in exon 8 or in exon 8A. –28 Tabl.p = 0.019) and more pts. were diagnosed in the first year of life .The detailed comparison of the groups is presented in Marked QTc prolongation (>500 ms) was present in all the patients, except two patients with exon 8 mutation. The degree of QTc prolongation (maximum QTc) was similar in the groups . There was no difference in the utilization of pacemaker/ICD implantation or of left cervical sympathectomy. MACE rate was high (67–83%) but was not different in the groups.The detailed comparison of the groups is presented in There was one additional family member with exon 8 mutation. His inclusion did not alter any of the statistical comparisons.There were 52 index patients with exon 8/8A and 33 index patients with non-exon 8/8A mutations. Exon 8/8A mutations almost all clustered at codon 402 (7 cases), 405 (2 cases), and 406 (42 cases), while the most frequently affected codons in non-exon 8/8A mutation carriers were codon 518 (three cases with p.Arg518Cys mutations and one case with p.Arg518His mutation), codon 857 (one case with p.Pro857Leu and p.Pro857Arg mutations each), codon 858 (four cases with p.Arg858His mutation), codon 1166  and codon 1473 (two cases with p.Ala1473Gly mutation) .The detailed comparison of the two groups is presented in p < 0.001). Extra-cardiac manifestations  were significantly more frequent in patients with exon 8/8A mutations. As TS was the overwhelmingly prevalent phenotype in patients with exon 8/8A mutations, the major phenotypic characteristics of TS were all significantly more frequent in patients with exon 8/8A mutations. QTc prolongation was present in all the 52 patients with exon 8/8A mutations, while it was seen in only in 79% of the patients with non-exon 8/8A mutations (p = 0.0025). The degree of QTc prolongation (maximum QTc) was much more pronounced in patients with exon 8/8A mutations  and the rate of pts. with >500 ms QTc prolongation was much higher . AV block was also observed in significantly more cases in patients with exon 8/8A mutations . There was no difference in the utilization of pacemaker/ICD implantation or of left cervical sympathectomy. There was a marked difference in terms of outcome, as much higher number of pts. with exon 8/8A mutations died  or experienced MACE .The predominant phenotype associated with exon 8/8A mutations was TS in 49 patients (96%), COTS in 1 patient (2%) and isolated LQT8 in 2 patient (2%), while with non-exon 8/8A mutations it was TS in 10 patients (29%), COTS in 5 patients (15%) and isolated LQT8 in 18 patients (56%) (Out of the 85 index patients there were 59 (69%) with TS, 6 (7%) with COTS and 20 (24%) with the isolated LQT8 phenotype.As detailed previously, exon 8 or 8A mutations, affecting codons 402, 405 and 406 made up the majority of the genotypes in patients with TS . Most mutations leading to COTS affected codon 518 in 4/6 patients (67%). Mutations causing isolated LQT8 scattered through the gene with only one codon having affected in more than one patient  .p < 0.001). In addition, significantly much more pts. with TS were diagnosed at birth or in the first year of life. The degree of QTc prolongation was much more prominent in patients with TS than in patients with COTS or with isolated LQT8  and the number of pts. with >500 ms QTc prolongation was much higher . There was no significant difference regarding PM/ICD/AED implantation or utilization of left cervical sympathectomy among the groups. There was a marked difference in terms of outcome, as a much higher number of pts. with TS died, as compared with COTS, or isolated LQT8  or experienced MACE .The detailed comparison of the three groups is presented in There was one additional family member with exon 8/8A mutations  and additional 48 family members with non-exon 8/8A mutations , and there was one additional family member with TS , 9 family members with COTS  and 39 family members with isolated LQT8 .As family members in every group usually exhibited a milder phenotype, all the statistical differences, observed between the index patient groups regarding disease and outcome parameters, remained statistically different, even at a higher significance level. Furthermore, the difference in age at death or at the time of MACE became statistically different, as patient with exon 8/8A mutations or patients with TS died at an earlier age and had MACE at an earlier age. Data are presented in CACNA1C gene mutation associated Timothy syndrome, “cardiac only” Timothy syndrome, isolated long QT syndrome 8 and provided data that these disease forms exhibit major differences regarding clinical manifestations and outcome. These differences can be defined either based on the genotype or the phenotype.In our systemic review we examined CACNA1C should be classified as TS1, and when the next TS disease gene is discovered, it can be classified as TS2  which is reflected in the similarly high MACE rate. All the above probably makes the distinction between TS1 and TS2 obsolete, at least with regard to clinical outcome, and may make the use of the descriptive terms “classical TS” (with syndactyly) and “non-classical TS” (without syndactyly) more appropriate, as it emphasizes that the two are similar diseases with comparable clinical outcomes.On the other hand, there are important differences between TS, COTS and isolated LQT8. Based on the phenotype, Timothy syndrome is characterized by a much earlier disease onset, much more pronounced QTc prolongation and much higher mortality than “cardiac only” Timothy syndrome or isolated LQT8. Although phenotypic differences are the basis for the categorization of these disease forms, this is the first time that differences in ECG parameters or in the clinical outcome have been demonstrated. The degree of QTc prolongation is often extreme in TS (>600 ms), and it is >500 ms in >90% of TS patients, while it is only mildly prolonged (<500 ms) in COTS or LQTS. This might be the primary reason for the higher rate of clinical complications, either death or MACE. Similar observations were made by Landstrom et al. based on 28 independent probands . Based oDifferences, like those outlined above, can be encountered if comparisons are made based on the genotype, i.e., between carriers of exon 8/8A mutations vs. non-exon 8/8A mutations. As exon 8/8A mutations are responsible for 83% of TS cases and only 17% of COTS and 10% of isolated LQT8, similar differences can be observed that was seen among TS, COTS and isolated LQT8. These include that disease onset is at much a younger age, phenotypic characteristics of TS are more prevalent, QTc prolongation is much more pronounced and clinical outcome is much more severe in patients with exon 8/8A mutations.Additionally, the results of our systemic review demonstrated that TS is a rather homogenous disease genetically, as the p.Gly406Arg mutation either in exon 8 or exon 8A alone is responsible for 70% of the cases, and mutations affecting codons 402–407 is responsible for 85% of TS cases. The strongest relationship was seen between mutation p.Gly406Arg in exon 8A and “classical” TS, which is present in 93.5% of the patients. COTS and isolated LQT8 are more heterogenous in this regard having their causative mutations more scattered through the gene.CACNA1C gene mutations alone are not sufficient to explain the above differences in phenotypic expression. CACNA1C mutations may result in channel dysfunction in different ways . One poogenesis .CACNA1C protein. The transcript profile of the CACNA1C gene was reported to be substantially more complex than appreciated by Clark et al., as they identified 38 novel exons and 241 novel transcripts (CACNA1C channels (Another explanation could involve the many different isoforms of the nscripts . Importachannels . A low lchannels .CACNA1C gene mutation associated Timothy syndrome, “cardiac only” Timothy syndrome, and isolated long QT syndrome 8 exhibit major differences regarding clinical manifestations and outcome. These differences can be defined either based on the genotype or on the phenotype. Among the phenotypes Timothy syndrome shows the most severe clinical manifestations with much earlier disease onset, much more pronounced QTc prolongation and a much higher mortality than “cardiac only” Timothy syndrome or isolated LQT8. However, distinguishing TS subtypes, in any form, are not supported by our data.This current comprehensive systematic review demonstrates that Implications for practice include that a high degree of surveillance is warranted if these diseases, especially TS is identified because of the high rate of adverse cardiac events. Genotyping of the patients help to stratify the risk of the patients as exon 8/8A mutations carry higher risk.Implications for research include the need for establishing a prospective world-wide registry to fully explore the phenotypic spectrum and clinical outcome of these diseases.This systematic review harbors all the intrinsic shortcomings of a retrospective study. Given the retrospective nature of the study, clinical information on every aspect of multiorgan involvement in the reported cases was not available. Although all efforts have been done to collect all the cases reported in the literature the study cohorts are still relatively small that limits our ability to draw definitive conclusions. Also, the timespan of the reports encompasses 15 years, from 2004 to 2019, which makes the comparison of individual reports difficult in terms of treatment and outcome over time.The original contributions presented in this study are included in the article/All authors listed have made a substantial, direct, intellectual contribution to the work, and approved it for publication.


Metabolic factors have been shown to be associated to severe radiographic knee osteoarthritis (RKOA). However, more knowledge is needed in early clinical knee osteoarthritis (KOA). The aim was to study associations between metabolic factors and radiographic knee osteoarthritis (OA) in individuals with knee pain. A second aim was to study associations between metabolic factors and RKOA in those with normal BMI and in those overweight/obese, respectively.This cross-sectional study included 282 individuals with knee pain (without cruciate ligament injury) and aged 30–67 years, and 70% women. Waist circumference, body mass index (BMI), proportion of fat and visceral fat area (VFA) were assessed. RKOA was defined as Ahlbäck grade 1 in at least one knee. Fasting blood samples were taken and triglycerides, cholesterol  and high density lipoprotein (HDL)), C-reactive protein (CRP), glucose, HbA1C were analysed. Metabolic syndrome was defined in accordance with the International Diabetes Federation (IDF). Associations were analysed by logistic regression.Individuals with RKOA were older, had higher BMI, higher VFA, larger waist circumference and had increased total cholesterol, triglycerides and LDL-cholesterol, but not fasting glucose. There was no difference between the group with RKOA vs. non-radiographic group regarding the presence of metabolic syndrome. In a subgroup analysis of individuals with normal BMI (n = 126), those with RKOA had higher VFA, more central obesity, higher levels of CRP and total cholesterol, compared with individuals without RKOA. In individuals with obesity, age was the only outcome associated to RKOA.There were clear associations between metabolic factors and RKOA in individuals with knee pain, also in those with normal BMI. In individuals with obesity age was the only variable associated to RKOA.clinicalTrials.gov Identifier: NCT04928170. Osteoarthritis (OA) is a group of diseases with various pathophysiological mechanisms, where metabolic, traumatic and age-related phenotypes seem to be significant , 2.The pathophysiological mechanisms in the metabolic phenotype involve factors that are mainly linked to obesity. Obesity affects the weight-bearing joints through increased load and chronic mechanical stress, which induces chondrocytes by mechanoreceptors to synthesize proinflammatory and cartilage-degrading mediators , 4. HoweMost studies of metabolic factors in knee OA (KOA) have been investigated in individuals with severe radiographic knee osteoarthritis RKOA. It is suggested that knee pain should be considered as the first sign of what eventually will become RKOA . The hypThe aim of this cross-sectional study was to investigate associations between metabolic factors and RKOA in individuals with knee pain. A second aim was to study associations between metabolic factors and RKOA in those with normal BMI and in those with obesity, respectively.This cross-sectional study present baseline data from an ongoing five-year longitudinal study, the Halland osteoarthritis cohort (HALLOA), n = 306. The HALLOA cohort profile has been published . The inc2; normal 18.5–24.9 kg/m2; overweight 25.0-29.9 kg/m2 and obese > 30.0 kg/m2). The two groups BMI < 25 kg/m2 and BMI ≥ 25 kg/m2, representing those under-/ normal weight as well as overweight/obese was also used. Proportion of fat and visceral fat area (VFA) were assessed by Inbody 770 (Biospace South Korea), which has an intercorrelation coefficient of 0.88-1.00, compared to dual-energy x-ray absorptiometry [2 [Waist circumference was manually assessed with a measuring tape (cm) around the waist, at the height of the navel. Central obesity was classified as waist circumference ≥ 94 cm in men and ≥ 80 cm in women . The heitiometry , 22. Raimetry [2 . Blood pmetry [2 .Fasting plasma glucose (mmol/L), triglycerides (TG) (mmol/L), total cholesterol (mmol/L), HDL-cholesterol and LDL-cholesterol (mmol/L), HbA1c (mmol/mol) and C-reactive protein (CRP) > 1.0 mg/L were measured from venous blood, in accordance with the current laboratory standards at Halland County Hospital in Halmstad, Sweden. CRP below 1.0 mg/L was further analysed with a sensitive CRP ELISA method (Abnova). Raised glucose was classified as fasting plasma glucose ≥ 5.6 mmol/L, or previously diagnosed type II diabetes . Raised The radiographs were obtained in a skyline view of patellofemoral (PF) joints, and posteroanterior radiographs of both tibiofemoral (TF) joints were obtained in weightbearing position with flexed knees. RKOA was defined in accordance with Ahlbäck , i.e. AhTo test the differences between groups, the Student’s t-test and the chi-squared test were used where appropriate. Spearman’s rank correlation coefficient was used to assess the relationships between continuous variables. The missing data have not been replaced. All missing data are addressed in the tables. The statistical power was estimated with known prevalence of MetS (14–50%) in previous OA studies, which will give a statistical power of 80% for detecting differences at a 5% significance level in this study. The significance tests were two-tailed and conducted at the 0.05 level of significance.The associations between outcome factors and RKOA were calculated by univariate logistic regression analyses. Variables associated with RKOA with a significance level of p ≤ 0.25 were separately included in a model adjusting for age and sex [Statistical analyses were performed using SPSS version 21.0 statistical software .A detailed description of the included individuals is shown in Table The group with RKOA (n = 63) were on average older, had higher VFA, central obesity, higher total cholesterol and LDL-cholesterol and were more likely to have raised TG, than the group without RKOA. No differences were seen for CRP levels, glucose levels and there was no difference in proportion of MetS between the groups, Table In a univariate logistic regression, age, BMI, VFA, waist circumference, central obesity, proportion of fat, raised TG and LDL-cholesterol were associated with RKOA, Table 2, n = 126), 16% had RKOA, 20% had raised fasting glucose, and 58% had central obesity. Those who had RKOA (n = 20) were older, had a higher rate of raised VFA, central obesity and total cholesterol, compared with those who did not fulfil the criteria for RKOA (n = 106), Table Of those with normal BMI  in this study group, as was to be expected. Obesity is a risk factor for knee OA [There was a high prevalence of individuals with central obesity, increased VFA and overweight/obese  . One thiAbout two thirds of the participants had raised blood pressure, with no differences between those with and without RKOA, which is in line with previous studies of individuals with radiological knee OA , 39. In One third of the individuals in the present study had raised fasting glucose levels, according to the IDF criteria; however, only one of the included individuals had treatment for diabetes (type II). The cut-off for raised fasting glucose in Sweden is 6.0 mol/L and the cut-off for raised fasting glucose in the IDF criteria is 5.6 mmol/L, which could explain why most of the participants were untreated for their raised fasting glucose. The mean value of fasting glucose in the present study is in the upper part of the normal range in Sweden (4.0–6.0 mmol/L). In the present study, fasting glucose levels were not associated with RKOA, as reported in other studies , 44. OneIn the group with normal BMI, those with RKOA had higher level of CRP. CRP was also associated with RKOA in the univariate model and showed a borderline significance in the multivariate logistic regression. The increased inflammation measured by serum CRP is probably caused both by synovial inflammation and inflammation due to obesity –48. The Lipids were increased in those with RKOA in the present study and total cholesterol, LDL-cholesterol and triglyceride levels were associated with RKOA. Dyslipidaemia has been reported in previous studies and has been suggested to be involved in the pathogenesis of OA , 12, 50.In individuals with overweight/obesity, 28% had RKOA. There were no differences in metabolic factors between the groups with and without RKOA. This finding could be explained by the fact that dyslipidaemia and diabetes type II are linked to obesity and both groups were overweight or obese , 52. BecOne fifth of participants in the present study had RKOA, and most had not had a radiographic examination of their knees before study start. The rareness of the studied cohort, still early in the disease course and with a high risk of developing knee OA, will be accentuated in longitudinal follow-up studies.A limitation in this study was that magnetic resonance imaging (MRI) was not assessed to evaluate the knees, which could have affected the result. The radiographs were scored according to Ahlbäck and not to Kellgren and Lawrence, which is more common. In this study, the aim was to get a cut off for RKOA and Ahlbäck grade I is comparable to Kellgren & Lawrence grade II . The useMetabolic factors were associated with RKOA, in this study, also in those with normal BMI. For individuals with normal BMI, increased load and mechanical stress is probably not the main cause of their RKOA; instead, metabolic factors could be of importance. The associations between metabolic risk factors and development of RKOA in individuals with knee pain, especially in those with normal BMI, needs to be assessed in longitudinal studies, with a focus on pathophysiological mechanisms.


Exercise may trigger bronchoconstriction, especially in a group of athletes in whom bronchospasm during exercise is reported to occur more frequently than in nonathletes. The aim of this study was to determine the prevalence and environmental risk factors contributing to exercise-induced bronchoconstriction (EIB) in adolescent athletes. A prospective study was conducted among a group of 101 adolescent athletes who underwent spirometry, exercise challenge, fractional exhaled nitric oxide (FeNO) measurements, and allergy assessment. The study group was divided into three subgroups of athletes based on the most common sports environments: swimmers, “indoor” athletes, and “outdoor” athletes. The clinical evaluation demonstrated a high frequency of EIB in the study group. Moreover, a large proportion of the athletes in whom EIB was observed reported no pre-existing symptoms suggestive of bronchospasm or asthma. Among patients without a previous diagnosis of asthma, clinical evaluation confirmed 22% with positive exercise challenges, compared with 77% of adolescents with negative test results. Moreover, among the athletes with a history of asthma, 39% had positive exercise challenges. Both EIB and asthma are common conditions that affect adolescent athletes. Physicians should pay particular attention to this group, as the symptoms can lead to under- and overdiagnosis. It is widely known that physical exercise has a beneficial effect on children’s development. Furthermore, a lack of physical activity can lead to asthma development, particularly in children . HoweverThe prevalence of EIB in adult athletes varies between 30% and 70% . Among tSeveral theories describe the development of bronchoconstriction during exercise. These include thermal, osmotic, neural, epithelial damage, and inflammatory theories. When analyzing the first two mechanisms, it is believed that, during exercise, there is an increase in evaporation and a “drying” of the airway mucosa. This leads to an ionic disturbance of the mucosal barrier and the release of inflammatory mediators from nearby epithelial cells, which subsequently lyse, causing a potentially strong bronchoconstrictive effect. Upon the termination of exercise activity, the subsequent warming of the respiratory tract leads to secondary hyperemia, increased mucus secretion, and the swelling of the mucous membranes. Parasympathetic fibers, which mainly innervate the respiratory tract, according to Gawlik et al., may also be stimulated by high-intensity exercise which contributes to increased muscle tone and the subsequent EIB development. Epithelial damage also affects the development of EIB, and this is often observed in athletes. Repeated hyperventilation, combined with the inhalation of harmful pollutants from the environment, has a significant detrimental impact on the airway epithelium. This can be especially observed in swimmers who are exposed to chlorine compounds or in endurance winter sports athletes exposed to cold air ,8.EIB is influenced by environmental factors, such as air temperature and humidity. Children living in urban environments are 1.6 times more likely to experience EIB compared with those living in rural areas . The higThis is a prospective study, conducted at the Department of Pediatrics and Allergology, at the Korczak Pediatric Center in Lodz, Poland, from March 2019 to January 2022. The study included a group of Polish Caucasian children who were athletes in sports schools and clubs located in the Lodz metropolitan area. Adolescents enrolled in the research were recruited to the study by a physician during a visit to a sports facility. Some of the participants were existing patients of our outpatient clinic. The following sports were eligible for the study: football, horse riding, tennis, dance, athletics, cycling, martial arts, gymnastics, floorball, basketball, volleyball, handball, and swimming. Enrolled patients trained a minimum of four times per week for approximately 90 min per day. Exclusion criteria included: diagnosis of chronic respiratory system disease other than asthma, lack of cooperation during the lung tests, and the presence of contraindications to performing the planned tests (set according to the Polish Society of Allergology). The study was approved by the Medical Ethical Committee of the Medical University of Lodz (No. RNN/303/17/KE).Each participant partook in two study visits. During the first visit, with written consent from parents/legal guardians and children, patients were qualified for the study. Participants were interviewed and examined by an on-site allergist. Caregivers filled a questionnaire requesting demographic data and past medical history. Children who received treatment for asthma were asked to withhold long-acting beta-2 mimetics (LABA) for a period of 24 h before the next visit. During the second visit, each patient received a skin prick test (or serum-specific immunoglobulin E (IgE) test), spirometry, pulmonary resistance test, a measurement of fractional exhaled nitric oxide (FeNO) concentration, and a standardized exercise challenge.All pulmonary function tests were performed using a Master Screen unit , as described elsewhere, in accordance with the American Thoracic Society and European Respiratory Society (ATS/ERS) guidelines . During An electronically controlled treadmill  was used for exercise tests on all athletes. During the challenge, the following parameters were monitored: heart rate, test room temperature (a requirement was set at less than 25 °C), and room humidity (less than 50%). Heart rate monitoring was performed by a pulse meter device, a built-in feature of the treadmill.The treadmill test lasted for 8 min with the incline of the treadmill set at 3°. The test consisted of a two-minute burn-up exercise  until stabilization of 95% of the calculated maximum heart rate was achieved. This speed was maintained for another 6 min, at which point the level of exercise was decreased. At 20 and 5 min before exercise, and at 1, 3, 6, 10, 15, and 20 min after exercise, a spirometry procedure identical to the aforementioned spirometry test was performed. The analyzed parameter was the percentage of FEV1. The exercise test was performed according to the recommendations for provocative tests in allergy by the ATS/ERS. The diagnosis of EIB was made on the basis of an FEV1 decrease from the baseline of equal to or more than 10% ,12. All 2O) was maintained by monitoring a visual display in order to eliminate contamination from nasal FeNO. Dead space and nasal FeNO  and FeNO from the lower respiratory tract  were automatically recorded by using the manufacturer’s software. Three FeNO measurements of the plateau phase were obtained, with less than 10% variation. The mean value of three successive, reproducible recordings was retained for statistical analysis. The recommendations used for the analysis were based on the study by Buchvold et al. [The fractional exhaled nitric oxide (FeNO) measurements were performed according to the ATS/ERS recommendations, with a chemiluminescence analyzer  and defined in parts per billion (ppb) . The anad et al. .Skin prick tests with airborne allergens  were performed on all patients. Alternatively, blood tests ) against airborne allergens were assessed. The material was obtained after sampling 10 mL of peripheral blood. The type of test depended on patients’ cooperation.At the end of the tests, each athlete underwent a physical examination. Two doses of salbutamol were administered if the patient exhibited cough, wheezing, or shortness of breath. Children who required further medical diagnosis and treatment were admitted to the hospital.p > 0.1 was considered to indicate normal distribution. The median instead of the mean was used for comparison in all cases in regard to distribution other than normal. Nonparametric tests were used for the skewed distributions. U Mann–Whitney and chi-squared tests with or without the Yates correction and the two-tailed exact Fischer’s test were applied to determine the differences between the two groups according to the type of data. A comparison between multiple groups was performed with a nonparametric analysis of variance ; if ANOVA yielded a significant difference, this was followed by between-group multiple comparisons with a nonparametric post hoc test. For all tests, p < 0.05 was deemed significant, although we also present results with 0.05 < p < 0.1 in the Kruskal–Wallis nonparametric analysis section.Statistical analyses were performed using Statistica software . The distribution of the data was assessed with the Shapiro–Wilk test, and In this study, 39 girls and 62 boys  adolescent athletes, aged 12–18 years, were included. There were no statistically significant results concerning EIB between the female and male gender. All the details of patient characteristics are shown in p = 0.04815). Moreover, in these groups, 21% of athletes from outdoor activities, 42% from indoor activities, and 14% of swimmers (p = 0.03605) had positive and very close-to-positive results for EIB. Prior to the study, 7 soccer players (18%) observed dyspnea during or after exercise, in comparison with 28 other patients (45%) (p = 0.00979). All the details concerning this division are included in When dividing patients into groups, namely athletes performing “outdoor” activities, those performing “indoor” activities, and swimmers , symptomp = 0.01548). A diagnosis of EIB was confirmed in 28% (n = 28) of adolescent athletes: 61% without asthma and 39% with asthma. All the data concerning asthma, symptoms, the diagnosis of EIB, and treatment are summarized in Asthma was diagnosed in 24 athletes. While analyzing individual sports, only four soccer players (10%) had a previous history of asthma (p = 0.0295). Furthermore, a similar pattern was observed for forced vital capacity (FVC) (p = 0.0257)  . Pulmonap = 0.0588). All the details concerning FeNO in athletes are included in Among all the examined athletes, 89 (88%) had significantly elevated fractional exhaled nitric oxide values. In this group, we did not detect the presence of atopy in 40 (45%) adolescents (p = 0.004021), trees (0.017704) (measurements were performed outside of the pollen season), and mold allergens (0.007743). In patients with a history of asthma, 4 athletes (10%) were found to have no atopy, compared with 20 athletes (33%) who were allergic to at least one allergen (p = 0.00898) and 17 (37%) with polyvalent atopy (0.00894). A similar analysis was performed for patients with positive exercise challenges without asthma: The results revealed that 12 athletes (30%) had no atopy, and 16 patients (26%) were allergic to at least one allergen; however, these results were not statistically significant (p = 0.85185).During the analysis of atopy in the patients, higher ΔFEV1 values were achieved by athletes diagnosed with atopy to grass , and nonallergenic factors  may affect the epithelium of the respiratory tract, potentially leading to the formation of EIB. The results of 40 nonatopic athletes in whom FeNO was elevated support the above statement. On this basis, we argue that nonallergic factors can also induce inflammation, including the well-known biomarker FeNO. This contradicts a large amount of scientific research, according to which higher FeNO levels are related to atopy. However, it is worth noting that FeNO levels are affected by other factors, such as age, sex, height, current smoking status, and diet .Although exercise is indicated in the prevention of asthma, it is emphasized that excessive airway stress can damage the airway epithelium, leading to chronic inflammation and bronchoconstriction . ParticuOur study revealed that indoor athletes achieved lower spirometry parameters compared with other athletes. Moreover, athletes practicing outdoor activities achieved better results; within this group, 21% had positive EIB results in comparison to 42% of indoor athletes. Additionally, a history of dyspnea during training was reported by 21% of outdoor athletes, compared with 47% of indoor athletes and 38% of swimmers.According to Ventura et al., the prevalence of asthma in soccer players was lower than the prevalence in other elite athletes . Their sVentura et al. suggested that the type of sport may influence the incidence of asthma, which has so far been observed more frequently in endurance athletes, e.g., long-distance runners or swimmers . It is cSome authors note the benefits of athletes completing questionnaires to help diagnose EIB. This approach comes with certain limitations, as athletes occasionally confuse EIB symptoms with the body’s normal response to exercise . BurnettOn the other hand, our study revealed that 45% of athletes who reported symptoms of EIB had a negative exercise challenge, among which 15% observed cough prior to study inclusion, and 27% reported dyspnea during or after exercise. As previous studies indicate, there is a need to consider alternative causes of EIB symptoms, such as exhaustion, exercise-induced laryngeal dysfunction, and exercise-induced hyperventilation . This waThe observation of the EIB phenomenon in athletes raises the question of who may develop asthma. The typical symptoms of EIB are wheezing, shortness of breath, cough, and tightness in the chest. Patients with asthma also experience such symptoms, raising the question of whether to consider an isolated case of EIB or asthma. According to the definition by the Joint Task Force on Practice Parameters in Allergy and Immunology, EIB is related to transient bronchoconstriction during or after exercise. EIB may or may not be related to asthma attacks, a relation previously termed exercise-induced asthma (EIA) . For thiAccording to Aggarwal et al., the prevalence of EIB in patients with asthma is approximately 90% . Our patFeNO measurement is considered to be an effective, noninvasive tool to assess eosinophilic airway inflammation. In our observations, this parameter reached the lowest values among outdoor athletes, compared with higher values in athletes training indoor activities. Among the examined adolescents, FeNO values for swimmers had intermediate results. It is important to remember that the pattern of inflammation in swimmers is neutrophilic; consequently, the role of FeNO in this group is ambiguous .In our research, FeNO levels in athletes with confirmed EIB were higher than those in adolescents in whom EIB was not yet confirmed. This result was not statistically significant, with a resulting smaller sample of respondents than initially assumed. According to Errson et al., among the group of EIB-confirmed adolescent athletes, FeNO was not related to the disease. This leads us to consider that the mechanisms of EIB in athletes and the general population are different .Our study revealed that the greater the decline in FEV1 during an exercise test in adolescent athletes, the greater the levels of atopy to grass, trees, and mold. According to Goossens et al., among young athletes, the prevalence of atopy is elevated . In a TuBased on the analysis of our collected data, we argue that, during exercise, factors such as inhaling cold air are the trigger for bronchospasm rather than airborne allergens. In the case of year-round mold allergens, one must consider whether these are responsible for inferior test results in indoor athletes.During our study, no reversibility test was performed to confirm asthma; this will be the subject of a future study. The less-than-optimal sample size of 101 patients was due to limitations incurred during the SARS-CoV2 pandemic. Another limitation was that patients on permanent medication were not reassessed for whether these existing treatments were indeed appropriate. Furthermore, the authors intended to identify environmental risk factors that promote EIB, but due to the pandemic, such opportunities were greatly restricted. Nonetheless, our study confidently analyzed the results from athletes in different disciplines and environments. We evaluated several factors that potentially contribute to the development of EIB. Our efforts seek to help physicians more properly diagnose suspected patients with EIB.Our research provided significant evidence that EIB is a common phenomenon among adolescent athletes. Our first “take-home” message is that not every athlete may report or be aware of symptoms of bronchospasm. This may lead to underdiagnosis and result in improper treatment. Secondly, we wish to draw attention to unsuitable conditions for adolescent athletes during training and highlight typical symptoms, such as cough and dyspnea. These factors may indicate EIB development. Our study concludes with two open questions: Could EIB-positive athletes develop asthma in the future? What treatment strategies should be employed in such patients?